Cell differentiation is the central characteristic of tissue morphogenesis which initiates during embryogenesis, and continues to various degrees throughout the life of an organism in adult tissue repair and regeneration mechanisms. The degree of morphogenesis in adult tissue varies among different tissues and is related, among other things, to the degree of cell turnover in a given tissue.
The cellular and molecular events which govern the stimulus for differentiation of cells is an area of intensive research. In the medical and veterinary fields, it is anticipated that discovery of the factor or factors which control cell differentiation and tissue morphogenesis will advance significantly the ability to repair and regenerate diseased or damaged mammalian tissues and organs. Particularly useful areas for human and veterinary therapeutics include reconstructive surgery, the treatment of tissue degenerative diseases including, for example, arthritis, emphysema, osteoporosis, cardiomyopathy, cirrhosis, degenerative nerve diseases, inflammatory diseases, and cancer, and in the regeneration of tissues, organs and limbs. In this and related applications, the terms "morphogenetic" and "morphogenic" are used interchangeably.
A number of different factors have been isolated in recent years which appear to play a role in cell differentiation. Recently, a distinct subfamily of the "superfamily" of structurally related proteins referred to in the art as the "transforming growth factor- .beta. (TGF-.beta.) superfamily of proteins" have been identified as true tissue morphogens.
The members of this distinct "subfamily" of true tissue morphogenic proteins share substantial amino acid sequence homology within their morphogenetically active C-terminal domains (at least 50% identity in the C-terminal 102 amino acid sequence), including a conserved six or seven cysteine skeleton, and share the in vivo activity of inducing tissue-specific morphogenesis in a variety of organs and tissues. The proteins apparently contact and interact with progenitor cells e.g., by binding suitable cell surface molecules, predisposing or otherwise stimulating the cells to proliferate and differentiate in a morphogenetically permissive environment. These morphogenic proteins are capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascularization, connective tissue formation, and nerve innervation as required by the naturally occurring tissue. The proteins have been shown to induce morphogenesis of both bone cartilage and bone, as well as periodontal tissues, dentin, liver, and neural tissue, including retinal tissue.
True tissue morphogenic proteins identified to date include proteins originally identified as bone inductive proteins. These include OP-1, (osteogenic protein-i, also referred to in related applications as "OP1"), its Drosophila homolog, 60A, with which it shares 69% identity in the C-terminal "seven cysteine" domain, and the related proteins OP-2 (also referred to in related applications as "OP2") and OP-3, both of which share approximately 65-75% identity with OP-1 in the C-terminal seven cysteine domain, as well as BMP5, BMP6 and its murine homolog, Vgr-1, all of which share greater than 85% identity with OP-1 in the C-terminal seven cysteine domain, and the BMP6 Xenopus homolog, Vgl, which shares approximately 57% identity with OP-1 in the C-terminal seven cysteine domain. Other bone inductive proteins include the CBMP2 proteins (also referred to in the art as BMP2 and BMP4) and their Drosophila homolog, DPP. Another tissue morphogenic protein is GDF-1 (from mouse). See, for example, PCT documents US92/01968 and US92/07358, the disclosures of which are incorporated herein by reference. Members of the BMP/OP subfamily and the amino acid sequence identities (expressed as percentages) between selected members of the TGF-.beta. superfamily are shown in FIG. 6.
As stated above, these true tissue morphogenic proteins are recognized in the art as a distinct subfamily of proteins different from other members of the TGF-.beta. superfamily in that they share a high degree of sequence identity in the C-terminal domain and in that the true tissue morphogenic proteins are able to induce, on their own, the full cascade of events that result in formation of functional tissue rather than merely inducing formation of fibrotic (scar) tissue. Specifically, members of the family of morphogenic proteins are capable of all of the following in a morphogenetically permissive environment: stimulating cell proliferation and cell differentiation, and supporting the growth and maintenance of differentiated cells. The morphogenic proteins apparently also may act as endocrine, paracrine or autocrine factors.
The morphogenic proteins are capable of significant species "crosstalk." That is, xenogenic (foreign species) homologs of these proteins can substitute for one another in functional activity. For example, dpp and 60A, two Drosophila proteins, can substitute for their mammalian homologs, BMP2/4 and OP-1, respectively, and induce endochondral bone formation at a non-bony site in a standard rat bone formation assay. Similarly, BMP2 has been shown to rescue a dpp.sup.- mutation in Drosophila. In their native form, however, the proteins appear to be tissue-specific, each protein typically being expressed in or provided to one or only a few tissues or, alternatively, expressed only at particular times during development. For example, GDF-1 appears to be expressed primarily in neural tissue, while OP-2 appears to be expressed at relatively high levels in early (e.g., 8-day) mouse embryos. The endogenous morphogens may be synthesized by the cells on which they act, by neighboring cells, or by cells of a distant tissue, the secreted protein being transported to the cells to be acted on.
A particularly potent tissue morphogenic protein is OP-1. This protein, and its xenogenic homologs, are expressed in a number of tissues, primarily in tissues of urogenital origin, as well as in bone, mammary and salivary gland tissue, reproductive tissues, and gastrointestinal tract tissue. It is expressed also in different tissues during embryogenesis, its presence coincident with the onset of morphogenesis of that tissue.
The morphogenic protein signal transduction across a cell membrane appears to occur as a result of specific binding interaction with one or more cell surface receptors. Recent studies on cell surface receptor binding of various members of the TGF-.beta. protein superfamily suggests that the ligands mediate their activity by interaction with two different receptors, referred to as Type I and Type II receptors to form a hetero-complex. A cell surface bound beta-glycan also may enhance the binding interaction. The Type I and Type II receptors are both serine/threonine kinases, and share similar structures: an intracellular domain that consists essentially of the kinase, a short, extended hydrophobic sequence sufficient to span the membrane one time, and an extracellular domain characterized by a high concentration of conserved cysteines.
Morphogenic proteins are disulfide-linked dimers which are expressed as large precursor polypeptide chains containing a hydrophobic signal sequence, a long and relatively poorly conserved N-terminal pro region of several hundred amino acids, a cleavage site and a mature domain comprising an N-terminal region which varies among the family members and a more highly conserved C-terminal region. The C-terminal region, which is present in the processed mature proteins of all known morphogen family members, contains approximately 100 amino acids with a characteristic motif having a conserved six or seven cysteine skeleton. Each of the morphogenic proteins isolated to date are dimeric structures wherein the monomer subunits are held together by non-covalent interactions or by one or more disulfide bonds. The morphogenic proteins are active as dimeric proteins but are inactive as individual monomer subunits.
As a result of their biological activities, significant effort has been directed toward the development of morphogen-based therapeutics for treating injured or diseased mammalian tissue, including, for example, therapeutic compositions for inducing regenerative healing of bone defects such as fractures, as well as therapeutic compositions for preserving or restoring healthy metabolic properties in diseased bone tissue, e.g., osteopenic bone tissue. Complete descriptions of efforts to develop and characterize morphogen-based therapeutics for non-chondrogenic tissue applications in mammals, particularly humans, are set forth, for example, in: EP 0575,555; WO93/04692; WO93/0575 1; WO94/06399; WO94/03200; WO94/06449; WO94/10203; and WO94/06420, the disclosures of each of which are incorporated herein by reference.
Certain difficulties may be experienced upon administration of naturally isolated or recombinantly produced morphogenic proteins to a mammal. These difficulties may include, for example, loss of morphogenic activity due to disassociation of the biologically active morphogen dimer into its inactive monomer subunits, and/or handling problems due to low solubility under physiological conditions.
Accordingly, a need remains for the identification of morphogen analogs, which mimic or enhance the physiological effects of a morphogenic protein, for example OP-1. The analogs may be modified, morphogenically active hOP-1 protein dimers, or fragments or truncated analogs thereof, peptides or small organic molecules. Preferably the analogs have enhanced therapeutic value, for example, by being more stable and/or more soluble under physiological conditions than naturally occurring hOP-1, or, for example, by having enhanced tissue targeting specificity, enhanced biodistribution or a reduced clearance rate in the body.
It is an object of the present invention to provide a database defining the atomic co-ordinates of the three-dimensional structure of mature hOP-1, all or a portion of which can be used as part of a computer system for designing and/or identifying a functional analog of hOP-1. Another object is to provide means for designing and/or identifying a molecule having enhanced solubility and/or stability under physiological conditions as compared with hOP-1 and which is capable of mimicking or enhancing the biological activity of hOP-1 in a mammal. Another object of the invention is to provide a therapeutic composition comprising an analog designed and/or identified, and produced by the methods of the invention, and suitable for administration to a mammal in need thereof, such as a mammal afflicted with a metabolic bone disease, e.g., a disease characterized by osteopenia. Another object of the invention is to provide methods and compositions useful for designing and/or identifying, and producing an hOP-1 antagonist capable of, for example, competing with hOP-1 for receptor binding, but incapable of inducing a receptor-mediated downstream biological effect.
These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.